Compositions and methods for isolating lung surfactant hydrophobic proteins SP-B and SP-C

ABSTRACT

The present invention describes compositions of biologically active surfactant proteins SP-B and SP-C of high purity and high-yield methods for isolating SP-B and SP-C.

INTRODUCTION

This invention relates to (1) a composition of matter in which thepolypeptide has the chemical structure, biophysical activity andphysiologic effects of the small, hydrophobic protein SP-B with a purityof ≧95%; (2) a second composition of matter in which the polypeptidesolute has the chemical structure, biophysical activity and physiologiceffects of the small, hydrophobic protein SP-C with a purity of ≧95%;and (3) the methods for producing the compositions of matter.

BACKGROUND OF THE INVENTION

Role of Surfactant in Pulmonary Physiology

Inhaled air containing oxygen travels through the trachea, the bronchi,and the bronchioles to the hundreds of millions of terminal alveoli. Theterminal alveoli are the air spaces in the lungs where oxygen is takenup by the blood in exchange for carbon dioxide.

At the interface between the gas in the terminal alveoli and the liquidof the lung tissue, (i) oxygen diffuses into the blood from the alveoliand (ii) carbon dioxide diffuses from the blood to the alveolar airbefore being exhaled. To diffuse from the alveolar gas to the blood, anoxygen molecule must traverse the liquid lining the alveoli, at leastone epithelial cell, the basement membrane, and at least one endothelialcell.

Pulmonary surfactant acts at the interface between alveolar gas and theliquid film lining the luminal surface of the cells of the terminalalveoli. The normal pulmonary surfactant lining is extremely thin,usually no more than 50 nm thick. Thus, the total fluid layer coveringthe 70 square meters of alveolar surface in an adult human is onlyapproximately 35 ml.

For materials to be effective lung surfactants, surfactant moleculesmust move rapidly to the surface of the liquid. Pulmonary surfactantfunctions by adsorbing to the surface of the liquid covering theselining cells and changing surface tension of the alveolar fluid duringthe respiratory cycle.

Surface tension is a characteristic of most liquid solutions. At theinterface between liquid and a gas phase, the movement of molecules atthe surface of the liquid is restricted by intermolecular forces actingon those molecules. The intermolecular forces have a net direction thattends to decrease the area of the surface. The net force at the surfaceis referred to as surface tension. Surface tension varies with molarity,temperature and multiple solutes. Surface tension has units of force perunit length (dynes/cm or mN/m). The vector of the surface tension forceis perpendicular to the plane of the interface.

The lungs of vertebrates contain surfactant, a complex mixture of lipidsand protein which causes surface tensions to rise during surfaceexpansion (inflation) and decrease during surface compression(deflation). During lung deflation, surfactant decreases surface tensionto ≦1 mN/m, so that there are no surface forces that would otherwisepromote alveolar collapse. Aerated alveoli that have not collapsedduring expiration permit continuous O₂ and CO₂ transport between bloodand alveolar gas and require much less force to inflate during thesubsequent inspiration.

In order to attain sufficient uptake of oxygen by the blood andexcretion of carbon dioxide from the blood, an animal's lungs mustventilate the terminal alveoli simultaneously and evenly. Eitherunsynchronized or uneven ventilation will prevent sufficient oxygenuptake into the circulating blood and result in the retention of carbondioxide in the body.

During inflation, lung surfactant increases surface tension as thealveolar surface area increases. A rising surface tension in expandingalveoli opposes over-inflation in those airspaces and tends to divertinspired air to less well-aerated alveoli, thereby facilitating evenlung aeration.

Surfactant Deficiency or Dysfunction

Although the exact composition and physical characteristics of naturallung surfactant have not been determined, material isolated from thelumen of lungs, termed natural surfactant, contains a mixture ofphospholipids, neutral lipids, and proteins. (lobe A, Ikegami M,Surfactant for the treatment of respiratory distress syndrome. Am RevRespir Dis, 1987; 136:1256-75.) The phospholipids are not specific tosurfactant, but are also present in other biologic materials,particularly membranes. The predominant phospholipids in surfactant,however, are disaturated phosphatidylcholines which are present in lowconcentrations in most membranes. Among the proteins found in the lunglumen are mucoproteins, plasma proteins, and lung specific proteins.

The alveoli are lined with epithelial cells that have a role inproducing surfactant, maintaining the activity of surfactant, andpreventing the inactivation of surfactant. The epithelial cells form acontinuous, tight barrier that normally prevents entry into the alveoliof molecules from the circulation that can inhibit surfactant.

The alveolar epithelium consists of at least two types of alveolarcells, referred to as type I and type II alveolar cells. The type IIalveolar cells normally synthesize both the phospholipids and proteinsthat are in lung surfactant, store newly synthesized material in theintracellular inclusion bodies, secrete the surfactant into the alveolarspace, absorb surfactant from the alveolar space, and metabolizematerial re-incorporated into the type II cell. The role of type I cellsin surfactant function has not yet been identified.

Lung surfactant is normally synthesized at a very low rate until thelast six weeks of fetal life. Human infants born more than six weeksbefore the normal term of a pregnancy have a high risk of being bornwith inadequate amounts of lung surfactant and inadequate rates ofsurfactant synthesis. The more prematurely an infant is born, the moresevere the surfactant deficiency is likely to be. Severe surfactantdeficiency can lead to respiratory failure within a few minutes or hoursof birth. The surfactant deficiency produces progressive collapse ofalveoli (atelectasis) because of the decreasing ability of the lung toexpand despite maximum inspiratory effort. As a result, inadequateamounts of oxygen reach the infant's blood.

Endogenous surfactant production typically accelerates after birth, evenin quite premature infants. If the infant survives the first few days,lung surfactant status generally becomes adequate.

Additional causes of respiratory failure from surfactant dysfunctionhave been reported due to defects in surfactant synthesis (congenitalprotein B deficiency), or in secretion or metabolism of surfactant(alveolar proteinosis). In addition, lung surfactant can be inhibitedand inactivated in vitro by a variety of proteins, cell wallphospholipids, enzymes, and other products of inflammatory responses.

Injury to juvenile and adult animals can also inactivate surfactant andproduce a respiratory failure with a similar pathophysiology to thesurfactant deficiency in premature infants. This respiratory failure isoften referred to as the Adult (or Acute) Respiratory Distress Syndrome,ARDS. This syndrome results from several simultaneous pathologicprocesses, one of which is generalized inhibition of the extra-cellularsurfactant in the alveolar space plus dysfunction of the type IIalveolar cells which adversely affect the synthesis, secretion, ormetabolism of surfactant.

Current treatment of respiratory failure includes supplementation ofoxygen, mechanical ventilation, and instillation or aerosolization ofmaterials with lung surfactant activity. Some patients die fromrespiratory failure despite current treatments, some survive withpermanently damaged lungs, and other patients recover after prolongedtherapy.

Hydrophobic Surfactant Proteins

Lung surfactants are complex materials composed of multiple moleculesthat interact physically, without combining chemically, to achieve theirbiologic activity. Natural lung surfactant contains lipids and proteins.There are two types of lung surfactant proteins, hydrophilic andhydrophobic. The two hydrophilic surfactant proteins identified to date,named SP-A and SP-D, are water soluble, chloroform insoluble,glycosolated, have polypeptide chains >25,000 Daltons and are notessential for lung surfactant activity at the air:gas interface. Thehydrophobic proteins, named SP-B and SP-C are not water soluble, arechloroform soluble, are not glycosolated, have polypeptide chains<26,000 Daltons after post translational modification to their activeform, and are essential for normal biophysical and biologic activity oflung surfactant.

All of these surfactant proteins have been sequenced. The SP-C sequencehas been determined in mice, dogs, rats, humans, cows and rats. Thehomology between the sequence of protein SP-C in humans and the sequenceof protein SP-C in other species is >80%. The SP-B protein sequence hasbeen determined for humans, dogs, rats, rabbits, mice and cows. Most ofthe sequence identity of SP-B is shared throughout species. (Whitsett JA and Baatz J E. Hydrophobic surfactant proteins SP-B and SP-C:molecular biology, structure and function in Robertson B, Van Golde L MG, Batenburg J J eds. Pulmonary Surfactant: From Molecular Biology toClinical Practice, Elsevier, N.Y., 1992, pp. 55-75.) Both SP-B and SP-Care synthesized initially as proproteins which comprise the activepolypeptide chain plus additional amino acids added to one or both ends.After synthesis, the proproteins of SP-B and SP-C are modified byproteolytic processes to remove the additional amino acids, therebyyielding the active molecule. This process is referred to aspost-translational modification.

Several forms of both the SP-B and SP-C active proteins are observednaturally. Monomers, single molecules, are observed as are oligomers orsmall numbers of chains bound together. Some oligomers are formed bysulfide bridges (ones that are broken into monomers by reducing agents)and some bound together by other, non-sulfide, bonds. Oligomers that areformed by sulfide bridges can be separated into monomers by reducingagents. It is unknown whether the proteins as synthesized or anyintermediates have biologic activity. It is also unknown whetherdifferences in activity exist between different oligomers, or betweenmonomers and oligomers of these proteins.

Hydrophobic Proteins in Lung Surfactant Drugs

Eight different surfactants have been developed to treat newborn infantswith Respiratory Distress Syndrome, RDS. Two lung surfactant drugs havelipids, but no surfactant proteins (Exosurf, ALEC). Three surfactantdrugs have lipids and significant amounts of SP-C but low levels of SP-B(Survanta, Surfacten, Curosurf). Three others have significant amountsof both hydrophobic surfactant proteins (Infasurf, Alveofact, bLES).Infasurf and Alveofact are more biophysically active than Survanta orCurosurf, which in turn are more biophysically active than Exosurf.(Hall S B, Venkitaram A R, Whitsett J A, et al.: Importance ofhydrophobic apoproteins as constituents of clinical exogenoussurfactants. Am Rev Respir Dis 1992; 145:24-30; Seeger W., Grube C.Gunther A. Schmidt R. Surfactant inhibition by plasma proteins:differential sensitivity of various surfactant preparations, Eur RespirJ 1993: 6:971-977.) Infasurf is more biologically active than Survanta,and Survanta is more biologically active than Exosurf, measured aseither biophysical or physiologic activity. (Cummings J J, Holm B A,Hudak M L, Hudak B B, Ferguson W H, Egan E A: Controlled clinicalcomparison of four different surfactant preparations insurfactant-deficient preterm lambs. Am Rev Respir Dis 1991;145:999-1003. Mizuno K, Ikegami M, Chen C M, Ueda T, Jobe A H.Surfactant protein-B supplementation improves in vivo function of amodified natural surfactant. Pediatr Res 1995; 37:271-276.) Clinically,Infasurf is more effective than Survanta or Exosurf, and Survanta ismore effective than Exosurf. (Hudak M L, Farrell E E, Rosenberg A A etal. A multicenter randomized masked comparison trial of natural versussynthetic surfactant for the treatment of respiratory distress syndrome.J Pediatr 1996; 128:396-406; Bloom B T, Kattwinkel J, Hall R T et al.Comparison of Infasurf (calf lung surfactant extract) to Survanta(beractant) in the treatment and prevention of RDS. Pediatrics in Press,July 1997; Hudak M L, Martin, D J, Egan, E A, et al. A multicenterrandomized masked comparison trial of synthetic surfactant versus calflung surfactant extract in the prevention of neonatal respiratorydistress syndrome. Pediatrics in press, July 1997.) The differences inactivity of lung surfactants are associated with the amount and type ofhydrophobic proteins they contain.

Preparation of Purified Hydrophobic Proteins

Surfactant proteins have been separated from surfactant lipids and SP-Bhas been separated from SP-C for academic investigations into proteinmetabolism and function. (Kogishi F, Kurozumi Y Fukite et al. Isolationand partial characterization of human low molecular weight proteinassociated with pulmonary surfactant. Am Rev Respir Dis 1988;137:1426-1431; Mathialagan N, Possmayer F. Low molecular-weighthydrophobic proteins from pulmonary surfactant. Biochem Biophys Acta1990: 1045:121-127; Takahshi A, Waring A J, Amirkhanian J, et al.Structure function relationships of bovine pulmonary surfactantproteins: SP-B and SP-C. Biochim Biophys Acta 1990, 1044:43-49; Wang Z,Gurel G, Baatz J E, Notter R H. Differential activity and lack ofsynergy of lung surfactant proteins SP-B and SP-C in interactions withphospholipids. J. Lipid Res. 1996; 37:1749-1760.) These methods describeorganic extraction of natural surfactant followed by single or multiplecolumn chromatography processes to separate hydrophobic proteins fromlipids. Separation of the two hydrophobic surfactant proteins from eachother has utilized repeated, additional column chromatography and/orpreparative SDS PAGE, and/or reverse phase HPLC. Evaluation of thepurity of the resulting proteins has been qualitative. Protein solutionsproduced by these methods have not reported yields. A process using onlydifferential solubility in organic solvents has been described, but themethod results in detectable levels of more than one polypeptide chainin the "pure" SP-B. (Beers M F, Bates S R, Fisher A B. Differentialextraction for the rapid purification of bovine surfactant protein B. AmJ Physiol 1992; 262:L773-L778.) None of these methods presents apractical method for securing significant quantities (milligrams orgrams per procedure) of hydrophobic surfactant proteins, i.e., (1) a≧50% yield of SP-B and/or SP-C from an amount of SP-B and/or SP-C in areference sample or (2) SP-B and/or SP-C of ≧95% purity frombiologically generated sources.

SUMMARY OF THE INVENTION

Definitions of Abbreviations

As used in this description, the following abbreviations have thefollowing meanings:

ARDS--Adult (or Acute) Respiratory Distress Syndrome

° C.--degrees celsius

cm--centimeter

D--Dalton

DPPC--dipalmatoylphosphatidylcholine

g--gravity

>--greater than

H₂ O--molecular abbreviation for water

HPLC--high pressure liquid chromatography

kD--kiloDalton

kg--kilogram

<--less than

l--liter

LPC--lysophosphatidylcholine

m--meter

μl--microliter

μm--micrometer (micron)

mg--milligram

min--minute

ml--milliliter

mm--millimeter

mM--milliMolar

mN--milliNewton

ng--nanogram

N--Newton

O₂ --molecular abbreviation for oxygen

PBS--pulsating bubble

PC--phosphatydilcholine

PC/DS--phosphatidylcholine--disaturated

PE--phosphatydilethanolamine

PG--phosophatidylglycerol

pH--negative logarithm of hydrogen ion concentration

PI--phosphatidylinositol

PL--phospholipid

RDS--Respiratory Distress Syndrome

rpm--revolutions per minute

SDS PAGE--sodium dodecylsulfate polyacrylimide gel electrophoresis

SP-A--surfactant protein A

SP-B--surfactant protein B

SP-C--surfactant protein C

SP-D--surfactant protein D

SPH--sphingomyelin

SN-1--position 1 of glycerol molecule

SN-2--position 2 of glycerol molecule

vol--volume

    ______________________________________                                        Symbols for Amino Acids:                                                      A          Ala        Alanine                                                                        B Asx Asparagine or aspartic acid                        C Cys Cysteine                                                                D Asp Aspartic acid                                                           E Glu Glutamic acid                                                           F Phe Phenylalanine                                                           G Gly Glycine                                                                 H His Histidine                                                               I Ile Isoleucine                                                              K Lys Lysine                                                                  L Leu Leucine                                                                 M Met Methionine                                                              N Asn Asparagine                                                              P Pro Proline                                                                 Q Gin Glutamine                                                               R Arg Arginine                                                                S Ser Serine                                                                  T Thr Threonine                                                               V Val Valine                                                                  W Trp Tryptophan                                                              Y Tyr Tyrosine                                                                Z Glx Glutamine or glutamic acid                                            ______________________________________                                    

New Compositions of Matter and Methods for Producing the Same.

The invention is a new composition of matter which is a purifiedpreparation of each of the SP-C and SP-B lung surfactant hydrophobicproteins which retain their full biophysical and physiologic activity.Each solution has ≧95% of peptides that are specific to a single proteinby N-terminal amino acid sequencing, total amino acid analysis and SDSpolyacrylamide gel electrophoresis. The new materials can be used tofabricate novel pharmacologic agents with specific SP-B and/or SP-Ccontents relative to other components.

According to another object of the present invention there is provided amethod for separating hydrophobic proteins SP-B and SP-C from aqueoussources containing other biologic molecules.

According to another object of the present invention there is provided amethod for separating hydrophobic proteins SP-B and SP-C from organicsolvent solutions containing other biologic molecules.

According to another object of the present invention, there is provideda high-yield method for separating SP-B and SP-C from biologicalsubstances.

Other objects and advantages of the present invention will be apparentto those skilled in the art.

DESCRIPTION OF THE FIGURES

SDS PAGE of Reduced and Un-reduced SP-C

FIG. 1 shows a 20% continuous SDS polyacrylamide gel of purifiedsurfactant protein SP-C run in Laemmli Buffer. All samples were appliedat the top of the gel. Proteins were visualized using Coomassie Blueoverlaid with a silver stain.

Lane 1=low molecular weight protein standards, 2.5, 6.2, 8.2, 14 and 17kD.

Lane 2=10 ng of un-reduced purified SP-C. SP-C is present as a dimer inthe ≈11 kD band and as a monomer in the ≈5 kD band. No other proteinband is present.

Lane 3=high molecular weight protein standards, 14, 20, 24, 29, 36, 45and 66 kD.

Lane 4=low molecular weight protein standards (see lane 1).

Lane 5=10 ng of purified SP-C with 5% mercaptoethanol to reducedisulfide bonds. Almost all the SP-C is now in the ≈5 kD band and only afaint trace remains un-reduced in the ≈11 kD band.

Lane 6=high molecular weight protein standards (see lane 3).

SDS PAGE of Reduced and Unreduced SP-B

FIG. 2 shows a 20% continuous SDS polyacrylamide gel of purifiedsurfactant protein SP-B run in Laemmli Buffer. All samples were appliedat the top of the gel. Proteins were visualized using Coomassie Blueoverlaid with a silver stain.

Lane 1=low molecular weight protein standards, 2.5, 6.2, 8.2, 14 and 17kD.

Lane 2=25 ng of un-reduced purified SP-B. SP-B is present primarily asan oligomer in a single band at ≈26 kD. Small traces are seen in bandsat ≈26 kD. Small traces are seen in bands at ≈14 kD and at ≈8 kD.

Lane 3=25 ng of purified SP-B with 5% mercaptoethanol to reducedisulfide bonds. All the SP-B is now in a ≈14 kD band and in a ≈8 kDband. The loss of definition of the bands is the result of overloadingprotein in the lane.

Lane 4=high molecular weight protein standards, 14, 20, 24, 29, 36, 45and 66 kD.

Deflation Pressure Volume Curves in Excised Rat Lungs

In FIG. 3, the volume of a freshly excised rat lung is plotted againstthe inflating pressure during a deflation cycle. Curve A is deflation inthe normal excised lungs after removal from a rat; Curve B is deflationin lung surfactant deficient lungs after 20 rinses with 0.9% saline.Surfactant deficient lungs have higher surface tension in expiration anddo not retain gas as well as normal lungs. In the region of the curve<20 cm H₂ O, the volumes in the deficient lung are much less than thosein the normal lung.

Curve 1 is deflation in surfactant deficient lungs treated by lungsurfactant lipids without any protein and is statistically identical tountreated surfactant deficient lungs. Curve 2 is lung deflation indeficient lungs treated by (lung surfactant lipids+1% SP-C, wt/wt).Lungs treated with this mixture retain gas significantly better thandeficient lungs, but still do not retain gas as well as normal lungs.Curve 3 is lung deflation in deficient lungs treated with (surfactantlipids+1% SP-C+0.75% SP-B, wt/wt/wt). When significant amounts of bothhydrophobic proteins are combined with surfactant lipids lung deflationis restored to normal in surfactant deficient lungs.

DETAILED DESCRIPTION OF METHOD OF PREPARATION OF PURIFIED PROTEINS SP-BAND SP-C

Sources of Proteins SP-B and SP-C

There are several sources of biologically produced active surfactantproteins appropriate for purification.

(a) Natural lung surfactant recovered from alveoli by lavage of the lunglumen with aqueous solutions or organic solvents.

(b) Lung tissue minced and/or pulverized.

(c) Tissue or secretions from transgenic animals which express both theproteins and proteolytic enzymes required for post-translationmodification of the pro-proteins so that active surfactant proteins arepresent in secretions (such as milk) or stored in tissues (such at fat).

(d) Active surfactant proteins produced by isolated cells of eithereukaryotic or prokaryotic origin in which the genes for production andprocessing of these proteins are expressed.

(e) Active surfactant proteins which are produced by in vitro processingof proproteins of SP-B and SP-C produced by biological processes.

Initial Preparation of Source Material

(a) If the source material is natural surfactant is recovered as asuspension in an aqueous liquid it is concentrated by centrifugal force.

(b) If the source material has surfactant proteins intermixed withbiologic products other than, or in addition to, natural surfactant, thesource material is homogenized in an aqueous solvent using physicalprocesses and separated from the solvent by centrifugal force.

Separation of Hydrophobic and Hydrophilic Components

The concentrated source material is then separated into hydrophilic andhydrophobic components by adding it to 4 parts of an aqueous solventcontaining halide salts (0.15M NaCl, for example), and then sequentiallymixing in (a) an organic solvent consisting of 5 parts non-polar solvent(chloroform, for example) and 10 parts polar organic solvent (methanol,for example), (b) another 5 parts of a non-polar organic solvent, (c)another 5 parts of the aqueous solvent, but without halide salts. (BlighE G, Dyer W J. A rapid method of total lipid extraction andpurification. Canad J. Biochem Physiol 1959; 37:911). After thoroughmixing the liquids separate into a non-polar layer containinghydrophobic elements, an interfacial zone suspended material and awater:polar solvent phase. The non-polar solvent phase is retained forfurther proceeding.

Separation of Lipids from Hydrophobic Proteins

The hydrophobic elements dissolved in a non-polar organic solvent arenow applied to a solid phase material for separation of molecules bysize and hydrophobicity such as chromatography column or separationgels. If chromatography is used (such as LH columns, Pharmacia, Uppsala,Sweden) the column solvent is a non-polar organic solvent:polar organicsolvent:acid mixture (chloroform:methanol:HCl, for example). If gels areused the hydrophobic components are recovered from the organic solventby evaporation and dissolved in an appropriate buffer and solubilizingagent (0.1% SDS, 192 mM glycine, 25 mM Tris at pH 8.3, for example).From the eluate of the separation devices, fractions are chosen thatcontain high levels of proteins and low levels of phospholipids. Thetotal phospholipid is measured by the method of Ames and protein by themethod of Peterson. (Ames B N. Assay of inorganic phosphate, totalphosphate and phosphatases. Method Enzymol 1966; 8:115-118; Peterson G.A simplification of the protein assay method of Lowry et al. which ismore generally applicable Analyt Biochem 1977; 83:346-56.) Thephospholipid:protein ratio is <1 (wt/wt) in every fraction appropriatefor further processing.

Separation of SP-B from SP-C

If the hydrophobic proteins are dissolved in any buffer containingsolvent, the buffers are removed by dialysis against the pure solvent.If the solvent in which the proteins are dissolved is not anon-polar:polar:acid mixture, the solvent is removed by evaporationunder vacuum and heat and the proteins are re-dissolved in anon-polar:polar:acid mixture solvent (chloroform:methanol:1N HCl,47.5:47.5:5, vol/vol, for example). To this mixture add an aqueoussolvent, (water in 33 parts, for example), polar organic solvent(methanol 47.5 parts, for example), polar organic solvent (chloroform47.5 parts, for example), and aqueous solvent (water 47.5 parts, forexample) are added sequentially. The mixture is allowed to separate intotwo phases, the organic solvent phase in which the SP-C is dissolved andthe polar solvent:aqueous phase in which the SP-B is dissolved. The twophases are separated and the content of the protein assayed by themethod of Peterson and the purity assayed by SDS PAGE.

EXAMPLES

Recovery from Biologic Milieu

Lung surfactant was harvested from the lungs of freshly slaughteredcalves by securing a plastic tube in the trachea of 20 excised lungs,instilling 0.15M NaCl through the tube into the lumen of the lung untilit was full and recovering the saline by applying suction. The procedurewas then repeated on each lung. The recovered saline was thencentrifuged at 14,000× G for 30 minutes at 5° C. The pellet was retainedand the supernatant was discarded.

The pellet was recovered and suspended in 0.15 M saline to a totalvolume of 400 ml. The 400 ml was combined with 1.5 liter of 1:2chloroform:methanol (vol/vol) and mixed by shaking. An additional 50 mlof chloroform was added and the mixed by shaking. Finally 500 ml ofdistilled water was added and mixed by shaking. The entire 2.4 literswas left to separate overnight at 4° C.

After 12 hours, the mixture had separated into a lower chloroform phase,an upper clear phase and an interphase of floccular material. Only thelower phase was recovered and the upper phase and interphase discarded.The chloroform was placed in a rotoevaporator and evaporated to drynessusing vacuum and a water bath at 99° C. The residual material wasresuspended in 100 ml of chloroform. Total phospholipid was assayed bythe method of Ames and total protein by Peterson's modified Lowrymethod.

An aliquot containing approximately 600 mg of phospholipids and 12 mg ofsurfactant apoprotein was applied to a 80 cm long 2.5 cm diameter LH 20column using a chloroform:methanol: 1N HCl, 47.5:47.5:5, vol/volsolvent. After the void volume, the first 4 fractions of 2.0 ml eachwere collected and retained. Analysis of these fractions revealed theycontained >60% of the protein applied to the column and that thephospholipid:protein ratio (wt/wt) in these fractions of the eluate was<1.

The 4 eluate fractions were pooled to contain 3.8 ml chloroform, 3.8 mlof methanol and 0.4 ml of 1N HCl. First 3.8 ml of methanol and 2.64 mlof water were mixed with the pooled eluates by shaking, then 3.8 ml ofchloroform was added and mixed by shaking, and finally, 3.8 ml of waterwas added and mixed by shaking. The resultant mixture was centrifuged at4,000× G for 15 minutes to separate the mixture into two phases, anupper methanol:water phase and slower chloroform phase. The upper phasewas carefully aspirated off the lower phase and both phases were storedseparately.

The upper phase was examined by SDS Page, N-terminal sequencing andamino acid analysis. SDS PAGE showed any SP-B (See FIG. 2). N-terminalsequencing and amino acid analysis was performed by AAI, an independentlaboratory. A single polypeptide chain was detected whose N-terminuswas: ##STR1## This sequence (SEQ ID NO:1) is identical to the sequencefor the first amino acids from the N-terminal region of bovine SP-B.(Olafson R W, Rink U, Kielland S, et al. Protein sequence analysisstudies on the low molecular weight hydrophobic proteins associated withbovine pulmonary surfactant. Biochem biophys Res Commun 1987;148:1406-1411.) Amino acid analysis used hydrolysis at 165° C. for 24hours in 6N HCl with 1% phenol and was consistent with pure SP-B. Theactual recovery of Leucine (L) was found to be 21% of all amino acids.Leucine is a well-recovered amino acid. If SP-C or another hydrophilicsurfactant protein had been present at 5% or greater of the total amountof protein, the recovery of Leucine would have been less than 19% of allamino acids expected.

The lower, chloroform phase of the final extraction was also examined bySDS PAGE, N-terminal sequencing and amino acid analysis using the samemethodologies as were used in analysis of the upper phase. SDS PAGE isshown in FIG. 1 and revealed only SP-C, and no SP-B. The N-terminalsequencing found a single polypeptide chain whose sequence was: ##STR2##This sequence (SEQ ID NO:2) is identical to the one published by Olafsonet al. for the N-terminal amino acids of bovine SP-C. Amino acidanalysis was consistent with pure SP-C. Complete digestion was notobtained, but none of the amino acids found in SP-B that are not foundin SP-C (i.e., Q, T, Y, S, F, W, H, E, or D) were detected insignificant amounts (≧2% of total amino acids) in the lower phase aminoacids analysis.

The activity of both the purified SP-B and the purified SP-C proteins isdemonstrated in FIG. 3. In FIG. 3, the volume of excised rat lungsduring deflation is graphed as a function of the decreasing distendingpressuring. Normal lungs are presented in Curve A and lung surfactantdeficient lungs in Curve B. The deficient lungs retain much less volumeat the same distending pressure than the normal lung once distendingpressure fall below 20 cm H₂ O. When surfactant lipids, without anyprotein are tested in deficient lungs (Curve 1) there is no improvementin deflation volume retention compared to the deficient lungs withoutany treatment. However, when the purified SP-C is added to surfactantlipids (Curve 2) there is improvement in the deflation volume retention,similar to that observed with SP-C containing, but SP-B deficientsurfactant drugs like Survanta (Hall et al. op cit). If both purifiedSP-C and purified SP-B are added to surfactant lipids (Curve 3) the fulldeflation volume retention is restored to deficient lungs.

Uses for Separated and Purified SP-B and SP-C

(a) Purified apoproteins can be used to investigate the specificbiophysical and biological functions of the individual proteins.

(b) Increasing the amount of SP-B or SP-C can improve the function ofsome current lung surfactant drugs. Purified proteins can be used toincrease the amount of hydrophobic proteins in such lung surfactantdrugs.

(c) Purified proteins can be used to develop standards for specificAssays of SP-B and SP-C.

(d) Purified proteins can be used as is, or after modification, asfunctional elements in new products that benefit the health of humansand animals.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 2                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: SP-B Peptide found in mammals                                  - - <400> SEQUENCE: 1                                                         - - Phe Pro Ile Pro Ile Pro Tyr Cys Trp Leu Le - #u Arg Thr Leu Ile           1               5 - #                 10 - #                 15              - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: SP-C Peptide found in mammals                                  - - <400> SEQUENCE: 2                                                         - - Leu Ile Pro Cys Cys Pro Val Asn Ile Lys Ar - #g Leu Leu Ile Val            1               5 - #                 10 - #                 15            __________________________________________________________________________

What is claimed:
 1. A composition of matter, comprising:(a) apolypeptide having an amino acid sequence; (b) wherein the amino acidsequence of the polypeptide comprises the amino acid sequence of SP-B;(c) wherein the polypeptide comprises at least 95% of the composition.2. A composition of matter, comprising:(a) a polypeptide having abiophysical activity; (b) wherein the biophysical activity of thepolypeptide corresponds to the biophysical activity of SP-B; (c) whereinthe polypeptide comprises at least 95% of the composition.
 3. Acomposition of matter, comprising:(a) a polypeptide having a physiologicactivity; (b) wherein the physiologic activity of the polypeptidecorrespond to the physiologic activity of SP-B; (c) wherein thepolypeptide comprises at least 95% of the composition.
 4. Thecomposition of claim 1, 2 or 3 wherein the polypeptide is produced froman in vivo process.
 5. The composition of claim 1, 2 or 3 wherein thepolypeptide is produced from a pro-protein that is produced by an invivo process.
 6. A composition of matter, comprising:(a) a polypetidehaving an amino acid sequence; (b) wherein the amino acid sequence ofthe polypetide comprises the amino acid sequence of SP-B; (c) whereinthe polypetide comprises at least 60% of the composition; (d) whereinthe composition is manufactured by a method in which the yield of thepolypeptide manufactured from an amount of the polypeptide in areference sample is at least 50%.
 7. A composition of matter,comprising:(a) a polypetide having a biophysical activity; (b) whereinthe biophysical activity of the polypetide corresponds to thebiophysical activity of SP-B; (c) wherein the polypetide comprises atleast 60% of the composition; (d) wherein the composition ismanufactured by a method in which the yield of the polypeptidemanufactured from an amount of the polypeptide in a reference sample isat least 50%.
 8. A composition of matter, comprising:(a) a polypetidehaving a physiologic activity; (b) wherein the physiologic activity ofthe polypetide corresponds to the physiologic activity of SP-B; (c)wherein the polypetide comprises at least 60% of the composition; (d)wherein the composition is manufactured by a method in which the yieldof the polypeptide manufactured from an amount of the polypeptide in areference sample is at least 50%.